Archivi del mese: gennaio 2014

Tessuti biomimetici su chip per il drug discovery

[Ghaemmaghami AM, Hancock MJ, Harrington H, Kaji H, Khademhosseini A. Biomimetic tissues on a chip for drug discovery. Drug Discov Today. 2012 Feb;17(3-4):173-81. doi: 10.1016/j.drudis.2011.10.029. Epub 2011 Nov 7.]

Figure 2

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273552/

Abstract:

Developing biologically relevant models of human tissues and organs is an important enabling step for disease modeling and drug discovery. Recent advances in tissue engineering, biomaterials and microfluidics have led to the development of microscale functional units of such models also referred to as ‘organs on a chip’. In this review, we provide an overview of key enabling technologies and highlight the wealth of recent work regarding on-chip tissue models. In addition, we discuss the current challenges and future directions of organ-on-chip development.

Nel testo:

A host of tissue models have been developed in academia and industry to mimic the sub-systems of organs or biological processes (Table 1 and Figure 2). In the following, we provide an overview of on-chip tissue models, their design, fabrication and physiological properties relevant to drug discovery. In particular, we highlight on-chip tissue models of the intestine, liver, lung and muscle, as well as models of tumors and blood vessels. We also outline recent attempts to combine the tissues of several organs on a single chip. Comprehensive reviews exist for bone, breast, cardiac, corneal, liver and tumor tissue models [5153] as well as neural networks [54,55]; we expand on this literature by focusing more on liver and tumor tissue models. Other on-chip models that could be used for future studies on drug discovery and toxicity include those for sensory organs such as the nose [41,56,57] and biological processes such as sperm motility [42,43,58] and wound healing [59,60].

Intestine on a chip

Orally administered drugs are mainly absorbed in the small intestine, where they must diffuse across a mucous layer covering an epithelial cell layer lining the intestinal wall [61]. Therefore, early in most development processes, drugs and chemicals are first screened against intestinal cells and tissues to assess their absorption, distribution, metabolism, elimination and toxicity (ADMET) [62]. The small intestine epithelium is composed of two main cell types: enterocytes and goblet cells. Enterocytes constitute ~90% of the cell population and are covered with surface-enhancing microvilli. In most areas of the intestine, goblet cells constitute the remaining 10% of the cell population and secrete mucus which coats the epithelial layer and is important for innate immune responses and homeostasis. Coordinated intestinal movements (i.e. peristalsis) have also been shown to have an important role in intestinal biology. Every material absorbed through the intestine into the blood stream must first diffuse across the mucus layer, the enterocytes, the lamina propria and the capillaries formed by endothelial cells. Crossing the epithelial layer, however, has been shown to be the rate-limiting step [63].

Small intestine models destined for absorption and/or metabolism studies should include these key cellular (i.e. enterocytes, goblet and vascular endothelial cells), structural (i.e. villi and mucus) and dynamic (i.e. peristalsis) features. Some of these elements were addressed by Kimura et al. [64], who engineered an intestinal model with a membrane and vascular flow simulating the epithelial barrier and the epithelial-endothelial interface, respectively. Additional important model features include the mucus layer and the digestion process. These were partially addressed by Mahler et al., who developed a microscale cell culture analog of the gastrointestinal (GI) tract incorporating digestion, a mucus layer and physiologically realistic cell populations [65]. To mimic drug transport across the intestinal wall better, additional structural features such as microscopic villi should be incorporated to enhance the intestinal epithelium surface area and to direct mucus. Such structural features have been fabricated in PDMS[2. AU: Please define] and hydrogels seeded with epithelial cells (Fig. 2j) [66], and could be incorporated into future functional tissue models.

Because healthy primary human cells are difficult to obtain, the majority of existing intestinal models were developed using immortalized cells lines. Although such cell lines can serve as informative and convenient surrogates for primary cells, their use limits the extrapolation of results to humans. Existing intestinal models also lack a host of ‘non-GI’ cells, including mast cells which play a key part in the barrier integrity and physiology of the intestinal epithelium [67].

Liver on a chip

Hepatotoxicity is a major side effect of many chemicals and drugs that are administered over prolonged periods of time. Almost half of all drug withdrawals occur as a result of acute liver toxicity [68]. Therefore, efficient, reliable, accurate and inexpensive tools for liver toxicity testing are required. Recent reviews provide an overview of microfluidic devices incorporating liver tissue or cells for metabolism, drug discovery and toxicity studies [53,69]. Existing tools for testing liver toxicity, involving hepatocyte cell lines, two-dimensional (2D) in vitro cultures and animal models, all suffer from major limitations. Cell lines and 2D cultur
es lack key metabolic activities found in human liver and major interspecies differences limit the predictive value of animal models [70,71]. The key challenge for on-chip liver tissue models is to maintain the metabolic activity and phenotype of the poorly viable cryopreserved human primary hepatocytes. Bioreactors have been developed to meet this challenge, such as the perfused multiwell plate device of Domansky et al. [38], which supported the growth and functional integrity of hepatocytes in 3D culture for up to a week (Fig. 2h), and the multiwell micropatterned co-culture system of Khetani and Bhatia [72] which could maintain phenotypic functions for several weeks. Both groups co-cultured primary hepatocytes with other key liver cells such as sinusoidal endothelial cells containing stellate and Kupffer cells [38] or fibroblasts [72] to improve and maintain primary hepatocyte function considerably. Another desirable feature of liver tissue models is to simulate the morphology of the liver’s functional units (i.e. lobules) to provide the homogenous and heterogeneous cell–cell interactions and cellular matrix support to maintain hepatocyte functionality [72,73]. Toh et al. [74] incorporated this feature into their on-chip liver model that exhibited a reasonable simulation of the liver morphology and functionality for up to 72 hours. In a second example, Schütte et al. used a perfusion system and integrated electrodes to assemble liver sinusoids by dielectrophoresis (Fig. 2g) [17]. A second class of on-chip liver models involves intact tissues extracted from humans or animals and integrated on-chip for the in vitro assessment of metabolism and toxicity [69]. Recently, van Midwoudet al. [75] integrated liver and intestinal slices into different compartments of a microfluidic device with sequential perfusion between the compartments to study interorgan interactions. In addition to reflecting in vivo conditions adequately for investigating liver drug metabolism and toxicity, on-chip models incorporating primary hepatocytes or intact tissue slices must be able to maintain key metabolic activities and normal liver integrity, particularly with regard to repeat dose experiments [69]. This feature was demonstrated by Khetani & Bhatia [72], who performed nine-day[3. AU: Is hyphen ok here? Added to draw a distinction between nine one-day repeat dose exps] repeat dose experiments with their co-culture system to test the chronic toxicity of troglitazone. Finally, an exciting new area is the ability to use in vitro cultures for disease modeling and to monitor disease progression and the impact of treatment in real-time. An example for such an approach is the recent work of Jones et al. [76], who used a fluorescent cell-based reporter system to monitor in real-time the infection of hepatitis C on hematoma cell lines and primary human hepatocytes.

Lung on a chip

The lung epithelium acts like a physical barrier between the host and the environment and responds to a variety of environmental insults such as pathogens and air pollution. The lung’s defense mechanisms include secreting antimicrobial peptides (e.g. defensins) and pro- and anti-inflammatory mediators, and recruiting innate and adaptive immune cells to the site of infection or damage. The efficiency of such protective responses as well as the homeostasis of the airways is underpinned by a variety of lung specialized epithelial cells (e.g. ciliated and mucus producing) and their crosstalk with other cell types such as antigen presenting cells, macrophages and myofibroblasts [77]. The human lung also provides one of the largest vascularized interfaces between the host and the environment, and is therefore considered a convenient port of delivery for local and systemic drugs. Drug delivery studies tend to focus on drug permeability across the lung epithelial barrier. Within this context, most existing lung tissue models are limited to single cell culture representations of the alveolar epithelium comprising epithelial cell lines [78,79], limiting their physiological relevance to the human lung. Certain enhancements have been made to single cell culture models, such as microfluidics and flexible membranes to apply fluid and solid mechanical stresses to single cell alveolar models to simulate the effects of mechanical ventilation and physiologic or pathologic liquid plug flows [8082].

More relevant lung tissue models destined for disease modeling and drug testing require additional cell types such as airway smooth muscle and immune cells, as well as key structural, functional and mechanical features of the human respiratory epithelium. Toward this goal, Huh et al. (Fig. 2b) [49] co-cultured alveolar epithelial, endothelial and certain immune cells on an artificial membrane and used adjacent vacuum channels to mimic the cyclic stretching during breathing. The inclusion of immune cells recapitulated certain aspects of human lung responses to infection and inflammation, enabling the assessment of the inflammatory response triggered by inhaled pathogens. Despite its many merits, this model used immortalized cell lines rather than human primary cells and did not include several other key structural or immune cells. It is not known whether the anatomical shape of the alveolar space or upper respiratory tract would have any bearing on the dynamics of cell–cell interaction. Future attempts, in addition to inclusion of primary cells, could focus on micropatterning scaffolds to improve the anatomical relevance and function of such tissue models.

Tumor on a chip

Developing anticancer therapies that target rapidly dividing cancer cells but leave normal healthy cells untouched is a grand challenge for cancer research. A recent review contains a comprehensive li
st of 3D tumor tissue models including multicellular spheroid, hollow fiber and multicellular layer models [53]. Perfusion delivers potential therapeutic agents to such 3D tumor cell cultures, which emulates the heterogeneous blood supply delivered to tumors in vivo [53]. In addition to these approaches, a popular strategy for developing anticancer drugs is HTS in which large combinatorial arrays of drug cocktails are produced and exposed to cells, which are then analyzed to assess the drug effects [31]. Performing HTS within microfluidic devices enables smaller volumes of reagents to be used than in typical multiwell plate experiments. One key engineering challenge is to generate an array of drug concentrations on-chip. To meet this challenge, Jang et al. developed a microfluidic combinatorial system that produced 64 or 100 combinations of two chemical solutions at different concentrations and stored them in isolated chambers [83]. The device inputs could be chosen such that the compositions of the stored mixtures spanned any triangle in the 2D space of chemical concentrations. A second engineering challenge is to expose different combinations or concentrations of drugs to cells cultured on-chip. Two groups recently accomplished this step by exposing human lung cancer cells to different concentrations of the anticancer drug verapamil (VP-16) [84,85]. In one study, the percentage of apoptotic cells in the verapamil-pretreated group was approximately double that of the control group, in agreement with previous flow cytometry analysis [85]. Further HTS advances could be made by using 3D cultures which mimic in vivo conditions better than cell monolayers. For example, Fischbach et al. [86] cultured cancer cells in 3D alginate hydrogels and found that cell signaling was altered.

A second line of cancer research is to test anticancer therapies on-chip to optimize system parameters for different types of cancer cells, while requiring minute amounts of reagents and cells. For example, Jedrych et al. [87] created a microfluidics system for in vitro photodynamic therapy (PDT) [87]. In PDT, light induces a photosensitizer delivered to the carcinoma cells, which in turn reacts with oxygen to produce a chemical toxic to the tumor cells. A key step in developing PDT is to optimize the system parameters such as the photosensitizer concentration for different types of cancer cells. Jedrych et al.[87] demonstrated this step by testing different concentrations of photosensitizer on A549 cancer cells.

Vessels on a chip

Blood vessels are involved in most medical conditions and are thus integral components to organs-on-chip devices [88]. A key engineering challenge is to grow blood vessels and capillaries on-chip. Dike et al. addressed this issue by using a surface patterned with 10 μm wide cell-adhesive lines surrounded by cell-repellent areas [89]. Endothelial cells cultured on the cell-adhesive lines differentiated to form capillary tube-like structures containing central lumens. A different approach was carried out by Kobayashi et al. [21], who patterned endothelial cells along parallel hydrophilic lines and then transferred the cells to a hydrogel scaffold. Once on the scaffold, the cells changed their morphology to form capillary-like tubular structures. The capillary structures were tested by flowing fluorescent solution through them, and also by transplanting them into mice where blood cells were subsequently found in the lumen. To control the endothelial capillary tube formation (tubulogenesis) further, Raghavan et al. cultured endothelial cells within microscale channels filled with collagen hydrogel (Fig. 2i) [90]. The cells were organized into tubes containing lumens with controlled branching and orientation. Future work must be done to generate capillary structures in vitro with similar mechanical properties to those in vivo.

A second major engineering challenge is to reproduce the microenvironment surrounding blood vessel walls, composed predominantly of vascular endothelial cells. A key component of this challenge is to understand how endothelial cells lining the vessel walls interact with other cells types. To address this issue, Chung et al. [91] and Kaji et al. [40,92] developed endothelium models to study the paracrine communication between endothelial and other cell types. Chung et al. found that MTLn3 cancer cells attracted endothelial cells to induce capillary formation, whereas smooth muscle cells suppressed endothelial activity [91]. Kaji et al. used complimentary substrates to co-culture endothelial and HeLa (cervical cancer) cells and found, similarly, that paracrine factors secreted from one cell type and directed via the culture medium to the other cell type affected the migration behavior of both types [40,92]. Research has also been devoted toward understanding how the cyclic stretch due to vessel deformation regulates cell function, and how disruptions in these forces produce diseased states such as hypertension or arteriosclerosis [93]. The effect of mechanical stimulation on endothelial cells in vitrohas been previously reviewed [94]. An additional challenge is to apply chemical and mechanical stimuli simultaneously to cells to engineer more biologically relevant models. To address this issue, Song et al. developed a membrane-based microfluidic device that models the adhesion of breast cancer cells on an endothelium (formed on the porous membrane) during intravascular metastasis (Fig. 2d) [95]. They showed that either the
addition of the chemokine CXCL12 from the basal side or the elevation of fluidic shear stress could enhance the metastatic cell adhesion. Many additional processes remain to be studied, including the chemical and cellular exchange across vessel walls, regulation of the permeability of surrounding tissues, coagulation of blood, transmigration of leukocytes and the regulation of vessel diameter.

Microfluidic platforms for probing the structure and function of actual blood vessels under physiological conditions also exist. Recently, Günther et al. developed a microfluidic platform enabling on-chip fixation, long-term culture, controlled delivery of drugs and automated acquisition of consecutive dose–response curves of intact mouse mesenteric artery segments (Fig. 2h) [96]. The phenylephrine dose–response relationships produced by the platform were in good agreement with those obtained by conventional pressure myography.

Muscle on a chip

Skeletal muscle is one of the major insulin-target tissues responsible for the maintenance of whole body glucose homeostasis. Defects in the glucose uptake in skeletal muscle contribute to insulin resistance in type 2 diabetes. To investigate insulin- and contraction-induced glucose metabolism, on-chip skeletal tissue muscle models require in-vivo-like structure, electrode stimulation and co-culture with primary human skeletal cells with motor neurons. In-vivo-like structural features include myotube alignment and assembly into sarcomeres. On-chip myotube alignment has been guided by substrate pattern [97,98] and stiffness [99,100] and by electrical stimulation [101]. An important aspect of a muscle tissue model is the ability to control muscle contraction. Such control has been accomplished by Tourovskaiaet al. [102] by stimulating myotubes with a laminar stream of agrin, a chemical secreted by neurons in vivo at the location where nerves contact the muscle. A different approach was developed by Kaji et al.[100], who modulated the substrate stiffness with an atelocollagen and electrically stimulated the formed myotubes to demonstrate a positive correlation between the contractility of the myotubes and the glucose uptake. Control of individual myotubes was achieved by Nagamine et al. [22], who integrated a microelectrode array with aligned myotubes on a fibrin gel sheet (Fig. 2e). Future improvements to on-chip muscle tissue models include monitoring the glucose uptake and other metabolism events in real time and incorporating neuromuscular junctions by co-culturing myotubes with neurons and stimulating them to induce muscle contraction.

Multiple organs on a chip

Most existing on-chip tissue models represent a single organ, preventing investigations on a drug’s systemic effect. Microscale cell culture analog systems, also known as ‘animal-on-a-chip’, ‘human-on-a-chip’ or ‘body-on-a-chip’ systems, attempt to improve the prediction of the effects of drugs and toxicity on the whole body. In a typical multiorgan device, multiple cell types representing different organs are cultured in separate chambers on a single chip. The chambers are connected by channels based on their sequence in vivo to assess systematically the effects of drug action and metabolism in different organs. A number of multiorgan devices has been developed with cell lines representing various organs or tissues, including: liver, tumor and bone marrow based on a mathematical pharmacokinetics–pharmacodynamics model and used to test the toxicity of the anticancer drug 5-fluorouracil (Fig. 2f) [103]; intestine, liver and breast cancer cells to evaluate the intestinal absorption, hepatic metabolism and the antitarget cell bioactivity of drugs [104]; liver, lung, kidney and adipose tissue [105]. Despite continued efforts and strong motivations to replace animal testing, these multiorgan systems are still in their infancy. The function of these systems is dictated by the cell types employed, and physiologically relevant cellular responses must be distinguished from artifacts due to in vitro cell culture. Also, barrier tissue analogs must be incorporated into existing multiorgan systems because these tissues can significantly reduce the bioavailability of drugs taken up through inhalation or absorbed through the skin. Finally, it is essential to benchmark the results of the on-chip systems with in vivo data under identical conditions and drug exposure. A more detailed discussion can be found in the literature by Eschet al. [106].

Annunci

Risposta ad “In Difesa della Sperimentazione Animale”

Oggi ci occuperemo di rispondere all’articolo “La Litania delle Reazioni Avverse” della pagina “In Difesa della Sperimentazione Animale”.

Essi iniziano citando diverse fonti non autorevoli su quanti effettivamente siano i dati dei morti per effetti avversi ai farmaci.
Facendo affidamento alle fonti ufficiali, scopriamo che il 51% dei farmaci commercializzati presenta gravi reazioni avverse che non si erano riscontrate in precedenza e che ogni anno gli effetti collaterali dei farmaci danneggiano 1,5 milioni di persone al punto da richiedere un’ospedalizzazione e 100 000 muoiono, questo solo negli USA [1].

Successivi studi hanno dimostrato che negli USA il numero dei morti per cause iatrogene è di 225 000 all’anno, di cui 106 000 non sono legate a errori nella prescrizione nè di altro tipo, il che ci conferma il dato allarmante [2].

I pro-SA affermano quindi:

[…] i farmaci non vengono commercializzati se prima non superano la fase clinica, condotta su pazienti umani. Pertanto, quando un farmaco viene ritirato dal mercato, ad aver “fallito” sono anche i software di simulazione e le colture in Vitro (i “metodi alternativi”) ed ha “fallito” persino la sperimentazione umana.

In primis, le metodologie alternative non si riducono solo alla sperimentazione su monocolture in vitro 2D, rimandiamo coloro che sono interessati al nostro elenco, diviso nei vari settori, sulle metodologie alternative, ricordando inoltre che la maggior parte di esse per essere sostitutiva dell’animale deve essere usata in maniera integrata con altre tecnologie alternative (la cosiddetta Integrated Testing Strategy o ITS).

In secondo luogo, come ci dice la stessa direttiva europea, “i tessuti e gli organi animali sono impiegati per lo svi­luppo di metodi in vitro”, il che significa che la maggior parte delle colture in vitro utilizza tessuto animale al posto di quello umano, il che ci porta ad includere i fallimenti di buona parte della sperimentazione in vitro in quelli della sperimentazione animale.

Terzo punto: certamente anche la sperimentazione clinica ha i suoi limiti, ma proprio per questo si sta cercando di ridurre le possibili minacce, e lo si sta facendo proprio migliorando la ricerca preclinica (o vogliamo forse pretendere di migliorare direttamente la ricerca clinica sull’uomo? Se sì, come?) e aprendo la strada alla medicina personalizzata.
Inoltre, ai fallimenti che si hanno dal passaggio dalla sperimentazione animale alla fase clinica I, si devono aggiungere quelli che si hanno nei trial clinici stessi e dopo la commercializzazione, per avere una visione globale di quanto la SA sia – alla fine dei conti – effettivamente efficace e di quali siano i suoi limiti.
In aggiunta, se già differenze genetiche intra-specifiche di appena lo 0,1% portano a grandi insuccessi, dovremmo capire che i risultati derivanti da specie che differiscono da noi per il 2 o il 15% di DNA hanno limitazioni ancora più ampie, che vanno al più presto risanate sostituendo i vecchi modelli con le metodologie avanzate.

Quarto ed ultimo punto, è risaputo che gli animali non prevedono l’81% dei gravi effetti avversi dei farmaci commercializzati. Gli stessi autori dello studio che ci fornisce questa cifra, ci spiegano come in realtà agli animali vengano somministrate dosi elevate e per lunghi periodi, proprio allo scopo di far emergere anche i potenziali effetti collaterali rari, quelli che potrebbero verificarsi proprio nei farmaci commercializzati. Riportiamo a tal proposito le parole degli autori, che ci fanno capire come nell’establishment tali studi siano considerati in grado di stabilire tali effetti avversi (nonostante i risultati che danno dimostrino l’opposto):

“One could argue that non-clinical studies are not designed to identify rare adverse reactions that appear after market approval.
Although the number of animals used in non-clinical studies is relatively small, the studies are designed to find important side effects that are likely to occur in humans (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2009). However, as high doses are administered for a prolonged period to elicit a complete toxicological response in animals, this approach can also estimate potential toxicities that could occur in humans.” 
[3]

Dunque ci chiediamo, se non servono a rilevare questi effetti avversi, perchè farli? E se dovrebbero farlo, perchè non considerarli fallimentari quando non riescono?

Per concludere, rimando i lettori al nostro elenco di articoli in cui confutiamo le teorie e i post dei gruppi come quello di cui abbiamo appena commentato l’articolo.

Bibliografia:

[1] Moore T.J., Psaty BM. e Furberg CD. Time to act on drug safety. JAMA, 279: 1571-1573, 1998.

[2] Starfield B. Is US health really the best in the world? JAMA. 2000 Jul 26;284(4):483-5. 

[3] van Meer PJ, Kooijman M, Gispen-de Wied CC, Moors EH, Schellekens H. The ability of animal studies to detect serious post marketing adverse events is limited. Regul Toxicol Pharmacol. 2012 Dec;64(3):345-9. doi: 10.1016/j.yrtph.2012.09.002. Epub 2012 Sep 12.

[A.C.]

Metodi alternativi per studi sulla tossicità per lo sviluppo: cellule staminali embrionali, tossicogenomica e CAESAR

[van Dartel DA, Piersma AH. The embryonic stem cell test combined with toxicogenomics as an alternative testing model for the assessment of developmental toxicity. Reprod Toxicol. 2011 Sep;32(2):235-44. doi: 10.1016/j.reprotox.2011.04.008. Epub 2011 May 6.]

Abstract:

One of the most studied in vitro alternative testing methods for identification of developmental toxicity is the embryonic stem cell test (EST). Although the EST has been formally validated, the applicability domain as well as the predictability of the model needs further study to allow successful implementation of the EST as an alternative testing method in regulatory toxicity testing. Genomics technologies have already provided a proof of principle of their value in identification of toxicants such as carcinogenic compounds. Also within the EST, gene expression profiling has shown its value in the identification of developmental toxicity and in the evaluation of factors critical for risk assessment, such as dose and time responses. It is expected that the implementation of genomics into the EST will provide a more detailed end point evaluation as compared to the classical morphological scoring of differentiation cultures. Therefore, genomics may contribute to improvement of the EST, both in terms of definition of its applicability domain as well as its predictive capacity. In the present review, we present the progress that has been made with regard to the prediction of developmental toxicity using the EST combined with transcriptomics. Furthermore, we discuss the developments of additional aspects required for further optimization of the EST, including kinetics, the use of human embryonic stem cells (ESC) and computational toxicology. Finally, the current and future use of the EST model for prediction of developmental toxicity in testing strategies and in regulatory toxicity evaluations is discussed.

[Cassano A, Manganaro A, Martin T, Young D, Piclin N, Pintore M, Bigoni D, Benfenati E. CAESAR models for developmental toxicity. Chem Cent J. 2010 Jul 29;4 Suppl 1:S4. doi: 10.1186/1752-153X-4-S1-S4.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913331/

Abstract:

BACKGROUND:

The new REACH legislation requires assessment of a large number of chemicals in the European market for several endpoints. Developmental toxicity is one of the most difficult endpoints to assess, on account of the complexity, length and costs of experiments. Following the encouragement of QSAR (in silico) methods provided in the REACH itself, the CAESAR project has developed several models.

RESULTS:

Two QSAR models for developmental toxicity have been developed, using different statistical/mathematical methods. Both models performed well. The first makes a classification based on a random forest algorithm, while the second is based on an adaptive fuzzy partition algorithm. The first model has been implemented and inserted into the CAESAR on-line application, which is java-based software that allows everyone to freely use the models.

CONCLUSIONS:

The CAESAR QSAR models have been developed with the aim to minimize false negatives in order to make them more usable for REACH. The CAESAR on-line application ensures that both industry and regulators can easily access and use the developmental toxicity model (as well as the models for the other four endpoints).

Test di teratogenesi usando cellule staminali pluripotenti umane

[Kameoka S, Babiarz J, Kolaja K, Chiao E. A high-throughput screen for teratogens using human pluripotent stem cells. Toxicol Sci. 2014 Jan;137(1):76-90. doi: 10.1093/toxsci/kft239. Epub 2013 Oct 23.]

Abstract:

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform’s utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.

Fegato bioartificiale per studi di epatotossicità, metabolismo, rigenerazione di epatociti, epatite virale, induzione enzimatica e trapianto

[Giri S, Braumann UD, Giri P, Acikgöz A, Scheibe P, Nieber K, Bader A. Nanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device. Int J Nanomedicine. 2013;8:1525-39. doi: 10.2147/IJN.S33589. Epub 2013 Apr 18.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632584/

Abstract:

Much effort has been directed towards the optimization of the capture of in vivo hepatocytes from their microenvironment. Some methods of capture include an ex vivo cellular model in a bioreactor based liver module, a micropatterned module, a microfluidic 3D chip, coated plates, and other innovative approaches for the functional maintenance of primary hepatocytes. However, none of the above methods meet US Food and Drug Administration (FDA) guidelines, which recommend and encourage that the duration of a toxicity assay of a drug should be a minimum of 14 days, to a maximum of 90 days for a general toxicity assay. Existing innovative reports have used undefined extracellular matrices like matrigel, rigid collagen, or serum supplementations, which are often problematic, unacceptable in preclinical and clinical applications, and can even interfere with experimental outcomes. We have overcome these challenges by using integrated nanostructured self-assembling peptides and a special combination of growth factors and cytokines to establish a proof of concept to mimic the in vivo hepatocyte microenvironment pattern in vitro for predicting the in vivo drug hepatotoxicity in a scalable bioartificial liver module. Hepatocyte functionality (albumin, urea) was measured at days 10, 30, 60, and 90 and we observed stable albumin secretion and urea function throughout the culture period. In parallel, drug metabolizing enzyme biomarkers such as ethoxyresorufin-O-deethylase, the methylthiazol tetrazolium test, and the lactate dehydrogenase test were carried out at days 10, 30, 60, and 90. We noticed excellent mitochondrial status and membrane stability at 90 days of culture. Since alpha glutathione S-transferase (GST) is highly sensitive and a specific marker of hepatocyte injury, we observed significantly low alpha GST levels on all measured days (10, 30, 60, and 90). Finally, we performed the image analysis of mitochondria-cultured hepatocytes at day 90 in different biophysical parameters using confocal microscopy. We applied an automatic algorithm-based method for 3D visualization to show the classic representation of the mitochondrial distribution in double hepatocytes. An automated morphological measurement was conducted on the mitochondrial distribution in the cultured hepatocytes. Our proof of concept of a scalable bioartificial liver modular device meets FDA guidelines and may function as an alternative model of animal experimentation for pharmacological and toxicological studies involving drug metabolism, enzyme induction, transplantation, viral hepatitis, hepatocyte regeneration, and can also be used in other existing bioreactor modules for long-term culture for up to 90 days or more.

Nel testo:

”Dopo 80 anni di dipendenza dalla sperimentazione animale che dà risultati imprevisti ed irrealistici, è il momento di esplorare il dispositivo modulare per sostituire gli animali e fornire risultati rilevanti per farmaci più sicuri […] Questa interazione 3D è molto efficace nel ricapitolare le funzioni fisiologiche degli epatociti in vivo dopo l’isolamento degli epatociti nel fegato in vivo. Le aziende farmaceutiche sono sotto pressione per sviluppare farmaci più sicuri perché un certo numero di farmaci approvati ed arrivati al mercato sono falliti negli ultimi dieci anni. Molte aziende farmaceutiche e biotecnologiche sono ora alla ricerca di strategie efficaci per migliorare la fase preclinica nel riconoscere farmaci candidati non selezionati all’inizio del processo di scoperta di nuovi farmaci. Le industrie farmaceutiche e biotecnologiche sono sempre più alla ricerca di un modello cellulare ex vivo che riproduca lo stato del fegato ad arte che possa sostituire l’uso di animali nei test di screening di farmaci nella fase iniziale di selezione dei candidati farmaci [ … ] Questo dispositivo modulare ottimizzato ha un grande potenziale nel predire la tossicità in vivo ed è molto adatto a misurare il metabolismo e la tossicità dei farmaci con una correlazione molto buona alla situazione in vivo. Esso minimizza il divario tra la situazione in vivo e quella in vitro. Questo studio proof of concept in un dispositivo modulare di fegato bioartificiale potrebbe sostituire milioni di animali che vengono attualmente sacrificati nei test preclinici ed aprire una nuova prospettiva per nuovi, più sicuri prodotti farmaceutici”

ReProTect: metodi alternativi per la tossicità riproduttiva

[Schenk B, Weimer M, Bremer S, van der Burg B, Cortvrindt R, Freyberger A, Lazzari G, Pellizzer C, Piersma A, Schäfer WR, Seiler A, Witters H, Schwarz M. The ReProTect Feasibility Study, a novel comprehensive in vitro approach to detect reproductive toxicants. Reprod Toxicol. 2010 Aug;30(1):200-18. doi: 10.1016/j.reprotox.2010.05.012. Epub 2010 May 21.]

Abstract:

ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the “Feasibility Study”, was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC(50) (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery. This comparative analysis together with a weight of evidence approach allowed a robust prediction of adverse effects on fertility and embryonic development of the 10 test chemicals in vivo. In summary, the vast majority of the predictions made based on the in vitro results turned out to be correct when compared to the whole animal data. The procedure used here, a nearest neighbor analysis coupled with a weight of evidence approach, may guide future activities in the field of alternative toxicity testing.

Differenze genetiche tra uomo e topo nella ricerca sulla distrofia muscolare

Uomini e topi: una piccola, grande differenza

Citiamo da Le Scienze:

Uomini e topi: una piccola, grande differenza

Due importanti caratteristiche di un gene chiave nello sviluppo della malattia sono presenti in quasi tutte le specie di mammiferi, uomo incluso, ma non nei topi e nei ratti

 

Uomini e topi hanno mostrato di avere differenze potenzialmente critiche, finora ignorate, in uno dei geni coinvolti nell’insorgenza della distrofia muscolare di Duchenne (DMD). A scoprirlo è stato un gruppo di ricercatori del King’s College di Londra che ne parlano in un articolo (Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat) pubblicato sulla rivista “BMC Biology”. 

In particolare hanno scoperto che due importanti caratteristiche di un gene chiave nella DMD sono presenti in quasi tutte le specie di mammiferi, uomo incluso, ma non nei topi e nei ratti. Questo risultato mette in questione il ricorso a questi animali come modello di riferimento per lo studio della malattia. 

La scoperta è stata fatta da Roland Roberts e collaboratori nel corso sello studio della alfa-distrobrevina, una sotto-unità citoplasmatica del complesso proteico associato alla distrofina, che nella DMD è disfunzionale.

La DMD è una miopatia che provoca la perdita di massa muscolare in tutto il corpo, ma può essere anche associata a effetti neurologici che possono manifestarsi come cecità notturna, disturbi nella visione dei colori o difficoltà di apprendimento. La α-distrobrevina è espressa in modo particolarmente spiccato proprio a livello cerebrale.

“Due differenze precedentemente non notate (un interruttore genico, o promotore, e un nuovo sito di legame per la sintrofina) sono codificate dal gene per la α-distrobrevina di quasi tutti i tetrapodi, eccetto che nel topo. Riteniamo che questo riconoscimento tardivo di caratteristiche chiave di un gene che è intensamente studiato fin dalla sua scoperta 13 anni fa sia dovuto al predominio del topo quale modello animale per lo studio della DMD e alla specifica distruzione di queste parti del gene nel topo”, ha osservato Roberts. 

Dal confronto con il genoma di altri roditori, risulta che questa semplificazione del gene per la alfa-distrobrevina nel topo e nel ratto si sia verificata fra i 30 e i 40 milioni di anni fa. (gg)

Riportiamo l’abstract dell’articolo in lingua originale:

[Böhm SV, Constantinou P, Tan S, Jin H, Roberts RG. Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat. BMC Biol. 2009 Dec 4;7:85. doi: 10.1186/1741-7007-7-85.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2796648/

Abstract:

BACKGROUND:

The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain) is its distant relative, alpha-dystrobrevin. The alpha-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands) in the dystrophin glycoprotein complex.

RESULTS:

We show here that, contrary to the literature, most alpha-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse alpha-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms alpha-dystrobrevin-4 and -5) are present in most mammals but specifically ablated in mouse and rat.

CONCLUSION:

Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the alpha-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the alpha-dystrobrevins (and therefore the dystrophin glycoprotein complexes) of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.